The present invention relates to an improved membrane based biosensor and to methods of improving the stability of membrane based biosensors.
Biosensors based on ion channels or ionophores contained within lipid membranes that are deposited onto conducting electrodes, where the ionophores are switched in the presence of analyte molecules have been described in International Patent Application Nos PCT/AU88/00273; PCT/AU89/00352; PCT/AU90/00025; and PCT/AU93/00509 (the disclosures of which are incorporated herein by way of reference). As these biosensors rely on changes in ion conduction through the membrane, usually mediated by an ionophore, it is important that there exists an ionic reservoir between the electrode and the lipid membrane. It is also important that the lipid membrane is at least in part linked to the reservoir and that the membrane is at least in part tethered to the conductive substrate. The stability of the substrate and the stability of the link between the reservoir and the substrate will influence the stability of the total membrane plus ionic reservoir system and thus the stability of the biosensor membrane.
The present inventors have now found that the stability of the biosensor membrane can be improved by incorporating a passivating layer between the reservoir region and the conducting substrate. This passivating layer serves to increase the bonding between the reservoir forming linkers and the conductive substrate as well as serving to protect the conductive substrate surface from corrosive or electrochemical effects of the aqueous solution. This would improve the stability of the biosensor both during storage and during the actual measurement of the biosensor response on addition of analyte containing sample. A reduction in the drift of the biosensor response during analyte addition is also obtained.
Generally, formation of the reservoir functionality onto conductive substrates is through binding of individual reservoir molecules with the substrate. The present inventors have determined that by introducing binding, passivating layer between the reservoir forming molecules and the electrode the stability of the whole membrane system is improved. There is also preferably binding between the molecules making up the passivating layer. This binding may include van der Waal""s forces, hydrogen bonding forces, ionic interactions or covalent linkage. Furthermore, if a thin, passivating layer is formed between the reservoir and the conductive surface such that water and ions are restricted from interacting with the conductive substrate then corrosive effects such as electrochemical degradation are minimised and the stability of the membrane is improved. This thin passivating layer may be eletrically insulating in nature. The ionic reservoir, in this case, has a reduced contact with the conductive substrate directly and information about the ion flux into or out of the ionic reservoir can be obtained by a variety of electronic transient pulse techniques commonly used to measure the charging and discharging of resistor/capacitor circuits.
Additionally, by introducing this thin layer between the ionic reservoir and the conductive substrate, then the spacing and orientation of the ionic reservoir forming molecules is determined by the structure of the thin layer substance rather than by the surface crystal orientation of the underlying conductive substrate.
Accordingly in a first aspect the present invention consists in a membrane based biosensor, the biosensor including an electrode, a passivating layer bound to the electrode, a lipid membrane incorporating ionophores, the conductivity of the membrane being dependent on the presence or absence of an analyte, an ionic reservoir between the membrane and the passivating layer, and spanning molecules spanning the ionic reservoir the molecules being covalently attached at one end to the membrane and at the other to the passivating layer.
In a preferred embodiment the biosensor includes a plurality molecules of following general structure:
Axe2x80x94Bxe2x80x94Cxe2x80x94D
in which:
A is a hydrophobic group of between 2-50 methylene units in length;
B is a group which spans the ionic reservoir;
C is a group capable of hydrogen bonding, forming van der Waal""s interactions, ionic bonding or covalent bonding with other molecules contained within the passivating layer; and
D is a group which binds to the conducting substrate.
In a preferred embodiment A is a hydrocarbon group of between 2-50 methylene units long, a phytanyl group, an unsaturated hydrocarbon of between 2-50 methylene groups long, a membrane spanning lipid, an archaebacterial lipid, a lipid hydrocarbon group, or a gramicidin derivative. It is presently preferred that A is a hydrocarbon group or an unsaturated hydrocarbon group of between 8-26 methylene units long or a gramicidin derivative.
In another preferred embodiment B is an oligoethylene glycol of between 4 to 20 ethylene glycol units long. Alteratively B may be repeating subunits of oligoethylene glycol of between 2 and 6 ethylene glycol units, the subunits being linked together via ester, amide or other linkages. It is highly preferred that in this arrangement that the linkages do not promote hydrogen bonding between the groups spanning the reservoir. In this regard it is preferred that the linkages are tertiary amides. As will be appreciated it is also highly preferred that linkages resistant to hydolysis such as tertiary amides are used.
In a further preferred embodiment group C of the above embodiment includes a secondary amide capable of hydrogen bonding with other amides; a hydrocarbon group capable of forming van der Waals interactions with other hydrocarbon groups, or a polymerisable group.
In a still further preferred embodiment D is a thiol, disulfide, sulfide, phosphine, silane or carboxylate.
It is further preferred that the group C in the above embodiment consists in a saturated hydrocarbon group of between 2 to 50 methylene units long, more preferably 8 to 30 methylene units long.
In a preferred embodiment the biosensor includes a plurality molecules of following general structure:
Cxe2x80x94D
in which C and D are as defined above.
As will be recognised in the biosensor of the present invention group A will be within and form part of the membrane, group B will span the ionic reservoir and groups C and D will be within and form part of the passivating layer.
In a preferred embodiment the biosensor has a structure as shown in FIG. 1.
In a further preferred embodiment the passivating layer has reduced permeability towards water and towards ions thus protecting the surface of the conductive substrate from destabilising effects due to water or ions.
In a preferred embodiment the biosensor includes a plurality molecules of following general structure:
Axe2x80x94Bxe2x80x94Cxe2x80x94D
in which:
A is a hydrophobic group such as a hydrocarbon group of between 2-50 methylene units long, a phytanyl group, an unsaturated hydrocarbon of between 2-50 methylene groups long, a membrane spanning lipid or archaebacterial lipid analog, a lipid hydrocarbon group, or a gramicidin derivative;
B is a group which spans the ionic reservoir;
C is a group that inhibits the permeation of water or ions to the conductive surface; and
D is a group capable of being attached to a conducting substrate such a a thiol, disulfide, sulfide, phosphine, silane, carboxylate or other group known to form strong interactions with surfaces.
In a preferred embodiment A is a hydrocarbon group or an unsaturated hydrocarbon group of between 8-26 methylene units long.
In another preferred embodiment B is an oligoethylene glycol of between 4 to 20 ethylene glycol units long. Alteratively B maybe repeating subunits of oligoethylene glycol of between 2 and 6 ethylene glycol units, the subunits being linked together via ester, amide or other linkages. It is highly preferred that in this arrangement that the linkages do not promote hydrogen bonding between the groups spanning the reservoir. In this regard it is preferred that the linkages are tertiary amides. As will be appreciated it is also highly preferred that linkages resistant to hydolysis such as tertiary amides are used.
In a further preferred embodiment group C includes a hydrocarbon group capable of forming van der Waals interactions with other hydrocarbon groups. The hydrocarbon group may contain a polymerisable group. Additionally the group may be functionalised at the distal end to the group D in order to modulate the function of the ion reservoir.
It is further preferred that the group C in the above embodiment comprises a saturated hydrocarbon group of between 2 to 50 methylene units long, more preferably between 8 to 30 methylene units long.
In a preferred embodiment the biosensor includes a plurality molecules of following general structure:
Cxe2x80x94D
in which C and D are as defined above.
In a yet another preferred embodiment the passivating layer is strongly bound to the conductive substrate and the functionality that make up the ionic reservoir.
Additionally it is preferred that the group C may be functionalised at the distal end to the group D. This modification may include the incorporation of hydrophilic or hydrophobic groups, amides, alcohols, acids, amines, acrylamide or acrylate or other polymerisable groups that may be used to further stabilise the passivating layer and may be used to modulate the ion reservoir function.
It is further preferred to use molecules such as those shown below and in FIGS. 2 and 3. 
where:
n=8 to 16
m=1 to 10
p=1 to 10
k=0 to 10
R, R1, R2, R3, R4=independently H, methyl, ethyl
L=hydrocarbon such as a phytanyl chain, or other lipidic hydrocarbon
P=a thiol, disulfide, sulfide, or other group for attaching to metal surfaces such as gold, platinum, palladium, silver etc; or a silane or alkoxy silane or chloro silane for attaching to silica or metal oxide or other conductive surface as is known to those skilled in the art.
In a second aspect the present invention consists in a biosensor of any aspects one or two wherein the flux of ions mediated by an ionophore through the membrane into the ionic reservoir is measured by transient electrical pulse techniques.